Project Summary Testicular germ cell tumors (TGCTs) are exceptionally sensitive to conventional genotoxic chemotherapy. This is likely due to the distinct DNA damage response (DDR) features of TGCTs and the germ cells from which they arise. Unlike somatic cells which often respond to DNA damage by arresting the cell cycle and conducting DNA repair, germ cells as well as long-lived pluripotent stem cells typically avoid the use of error-prone repair mechanisms and favor apoptosis, reducing the risk of genetic alterations in subsequent generations. Similarly, the TGCT precursor lesion, germ cell neoplasia in situ, does not show activation of a DDR, whereas precursor lesions of most somatic cancers express markers of an activated DDR in response to oncogene activation which serves as a barrier to tumor progression. To study TGCTs, our lab has developed the first genetically engineered mouse model of malignant TGCTs by conditional activation of Kras, an oncogene, and inactivation of Pten, a tumor suppressor gene, in germ cells. The malignant teratocarcinomas generated in these mice are composed of pluripotent embryonal carcinoma (EC) and differentiated teratoma tissue. Interestingly, EC cells, both in vivo and cultured in vitro, express stem cell markers, have tumor propagating activity, and are readily killed following chemotherapy treatment. Using cells derived from this model, the experiments proposed here will elucidate the DDR properties of TGCTs and the cells from which they arise, which will inform the mechanisms underlying their exceptional chemosensitivity. Specifically, this proposal aims to: understand how the cells that give rise to TGCTs respond to oncogenic events, apparently avoiding DDR activation (Aim 1), and determine the mechanism underlying the chemosensitivity of the embryonal carcinoma components of TGCTs and the chemoresistance of their differentiated counterparts (Aim 2). Aim 1 will be investigated by generating primordial germ cell-like cells (PGCLCs) and embryonic germ cell-like cells (EGCLCs) from induced pluripotent stem cells (iPSCs) derived from mouse embryonic fibroblasts (MEFs) with conditional Pten and Kras alleles and assessing malignant transformation, the degree of DNA replication stress, the extent of DNA damage, and the nature of the DDR in untransformed and transformed cells. For Aim 2, I will analyze differential gene expression in EC and differentiated cells before and after treatment with genotoxic chemotherapy and investigate the role of differentially regulated pathways in the chemosensitivity phenotype. It is critically important to study this curable cancer because understanding the basis of TGCT chemosensitivity will apply broadly to the development of treatments for the many other cancers that do not respond favorably to conventional chemotherapy. Additionally, understanding the malignant transformation of pluripotent cell types, including embryonic germ cells and iPSCs, will be important for the improvement of stem-cell based therapies, which carry the risk of tumorgenicity.